Department for Internal Medicine, Endocrinology and Nephrology, University of Leipzig, Leipzig, Germany.
Clin Endocrinol (Oxf). 2010 Dec;73(6):815-20. doi: 10.1111/j.1365-2265.2010.03872.x.
Constitutively activating mutations (CAMs) of the TSHR are the major cause for nonautoimmune hyperthyroidism. Re-examination of constitutive activity previously determined in CHO cell lines recently demonstrated the caveats for the in vitro determination of constitutive TSHR activity, which leads to false positive conclusions regarding the molecular origin of hyperthyroidism or hot thyroid carcinomas.
Mutations L677V and T620I identified in hot thyroid carcinomas were previously characterized in CHO and in 3T3-Vill cell lines, respectively, stably expressing the mutant without determination of TSHR expression. F666L identified in a patient with hot thyroid nodules, I691F in a family with nonautoimmune hyperthyroidism and F631I identified in a hot thyroid carcinoma were not characterized for their in vitro function. Therefore, we decided to (re)evaluate the in vitro function of these five TSHR variants by determination of cell surface expression, and intracellular cAMP and inositol phosphate levels and performed additionally linear regression analyses to determine basal activity independently from the mutant's cell surface expression in COS-7 and HEK(GT) cells.
Only one (F631I) of the five investigated TSHR variants displayed constitutive activity for G(α) s signalling and showed correlation with the clinical phenotype. The previous false classification of T620I and L677V as CAMs is most likely related to the fact that both mutations were characterized in cell lines stably expressing the mutated receptor construct without assessing the respective receptor number per cell. Other molecular aetiologies for the nonautoimmune hyperthyroidism and/or hot thyroid carcinomas in these three patients and one family should be elucidated.
促甲状腺激素受体（TSHR）的组成性激活突变（CAM）是导致非自身免疫性甲状腺功能亢进的主要原因。最近对先前在 CHO 细胞系中确定的组成活性进行了重新检测，这表明体外测定 TSHR 活性存在局限性，这导致了关于甲状腺功能亢进或热甲状腺癌分子起源的错误阳性结论。
先前在 HOT 甲状腺癌中鉴定的突变 L677V 和 T620I 分别在 CHO 和 3T3-Vill 细胞系中进行了特征鉴定，这些细胞系稳定表达突变体而未测定 TSHR 表达。在 HOT 甲状腺结节患者中发现的 F666L、在非自身免疫性甲状腺功能亢进家族中发现的 I691F 和在 HOT 甲状腺癌中发现的 F631I 均未对其体外功能进行特征鉴定。因此，我们决定（重新）评估这五个 TSHR 变体的体外功能，通过测定细胞表面表达、细胞内 cAMP 和肌醇磷酸盐水平，并在 COS-7 和 HEK(GT)细胞中进行额外的线性回归分析，以独立于突变体的细胞表面表达来确定基础活性。
在所研究的五个 TSHR 变体中，只有一个（F631I）显示出 G(α)s 信号的组成性活性，并且与临床表型相关。先前将 T620I 和 L677V 错误地归类为 CAM，最可能与以下事实有关，即这两种突变均在稳定表达突变受体构建体的细胞系中进行了特征鉴定，而没有评估每个细胞的受体数量。在这三个患者和一个家族中，应该阐明其他非自身免疫性甲状腺功能亢进和/或 HOT 甲状腺癌的分子病因。
